U.S. patent application number 16/604599 was filed with the patent office on 2020-09-24 for compounds, composition and uses thereof for treating cancer.
The applicant listed for this patent is INSTITUT GUSTAVE ROUSSY. Invention is credited to MOUAD ALAMI, SEBASTIEN APCHER, MATHILDE BOULPICANTE, ROMAIN DARRIGRAND, RENKO ZAFIARISOA DOLOR, ALISON PIERSON.
Application Number | 20200297741 16/604599 |
Document ID | / |
Family ID | 1000004901231 |
Filed Date | 2020-09-24 |
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United States Patent
Application |
20200297741 |
Kind Code |
A1 |
APCHER; SEBASTIEN ; et
al. |
September 24, 2020 |
COMPOUNDS, COMPOSITION AND USES THEREOF FOR TREATING CANCER
Abstract
The present invention relates to the fields of medicine and
cancer treatment. The invention more specifically relates to new
compounds which are typically for use as a medicament. In
particular, the invention relates to the use of these new compounds
for increasing the presentation, typically the production and
presentation, of Pioneer Translation Products (PTPs)-derived
antigens by cancer cells in a subject, and inducing or stimulating
an immune response in the subject. The present disclosure also
relates to uses of such compounds, in particular to prepare a
pharmaceutical composition and/or to allow or improve the
efficiency of a cancer therapy in a subject in need thereof. The
invention also discloses methods for preventing or treating cancer,
cancer metastasis and/or cancer recurrence in a subject. The
present invention in addition provides kits suitable for preparing
a composition according to the present invention and/or for
implementing the herein described methods.
Inventors: |
APCHER; SEBASTIEN;
(FRANCONVILLE, FR) ; PIERSON; ALISON; (VILLEJUIF,
FR) ; BOULPICANTE; MATHILDE; (CHEVILLY-LARUE, FR)
; DOLOR; RENKO ZAFIARISOA; (LES ULIS, FR) ; ALAMI;
MOUAD; (BUSSY SAINT GEORGES, FR) ; DARRIGRAND;
ROMAIN; (IVRY SUR SEINE, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT GUSTAVE ROUSSY |
VILLEJUIF |
|
FR |
|
|
Family ID: |
1000004901231 |
Appl. No.: |
16/604599 |
Filed: |
April 11, 2018 |
PCT Filed: |
April 11, 2018 |
PCT NO: |
PCT/EP2018/059213 |
371 Date: |
October 11, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/665 20130101;
A61K 31/352 20130101; A61P 35/00 20180101; A61K 45/06 20130101 |
International
Class: |
A61K 31/665 20060101
A61K031/665; A61K 45/06 20060101 A61K045/06; A61P 35/00 20060101
A61P035/00; A61K 31/352 20060101 A61K031/352 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 12, 2017 |
EP |
17305438.8 |
Oct 23, 2017 |
EP |
17306456.9 |
Claims
1-14. (canceled)
15. A medicament comprising a compound of formula ##STR00010##
wherein R.sup.1 and R.sup.2 are independently selected from the
group consisting of Na, H, --CH.sub.3, --CH.sub.2--CH.sub.3,
--CH.sub.2--CH.dbd.CH.sub.3, n-CH.sub.2--CH.sub.2--CH.sub.3,
P(O)(O--CH.sub.2--CH.sub.3).sub.2, P(O)(OH).sub.2 or
P(O)(ONa).sub.2 and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and
R.sup.5 are not H simultaneously, wherein R.sup.1 is not --CH.sub.3
when R.sup.2 is P(O)(ONa).sub.2 or P(O)(OH).sub.2 and each of
R.sup.3, R.sup.4 and R.sup.5 is H, and wherein R.sup.1 is not
--CH.sub.3 or H when R.sup.2 is --CH.sub.3 and each of R.sup.3,
R.sup.4 and R.sup.5 is H; and wherein R.sup.3, R.sup.4 and R.sup.5
are independently selected from the group consisting of H,
CH.sub.3, --CH.sub.2--CH.sub.3, --CH.sub.2--CH.dbd.CH.sub.3, and
C.sub.nH.sub.2n+1 with n=3-10.
16. The medicament according to claim 15, wherein the compound is
##STR00011##
17. A method for treating cancer, cancer metastasis and/or cancer
recurrence in a subject in need thereof, wherein the method
comprises a step of administering the subject with an effective
amount of a compound as described in claim 15.
18. The method according to claim 17, wherein the compound is
administered to the subject in combination with an effective amount
of at least one distinct anticancer agent, and/or the method
further comprises a step of exposing the subject to
radiotherapy.
19. The method according to claim 17, wherein cancer is selected
from the group consisting of carcinoma, sarcoma, lymphoma, germ
cell tumor, blastoma, leukemia and multiple myeloma.
20. The method according to claim 19, wherein the carcinoma is a
melanoma, a lung cancer or a breast cancer.
21. The method according to claim 18, wherein the at least one
distinct anticancer agent is selected from the group consisting of
a chemotherapeutic agent, an immune checkpoint blocker and an
anti-cancer vaccine.
22. The method according to claim 17, wherein the method is for
stimulating an anti-cancer immune response in a subject in need
thereof.
23. The method according to claim 17, wherein the subject is a
mammal.
24. The method according to claim 23, wherein the subject is a
human being.
25. The method according to claim 17, wherein the compound is
##STR00012##
26. A composition comprising a compound of formula (I) as described
in claim 15 and a pharmaceutically acceptable carrier.
27. The composition according to claim 26, wherein the composition
further comprises at least one distinct anticancer agent to be used
simultaneously, separately or sequentially.
28. The composition according to claim 26, wherein the compound is
##STR00013##
29. A method for inducing or increasing the presentation of Pioneer
Translation Products (PTPs)-derived antigens by cancer cells in a
subject, wherein the method comprises a step of administering the
subject with an effective amount of a compound as described in
claim 15.
30. The method according to claim 29, wherein the subject is a
mammal.
31. The method according to claim 30, wherein the subject is a
human being.
32. The method according to claim 29, wherein the compound is
##STR00014##
33. A kit comprising the compound of formula (I) as described in
claim 15, and at least one distinct anticancer agent in distinct
containers.
Description
FIELD OF THE INVENTION
[0001] The present disclosure generally relates to the fields of
medicine and cancer treatment. The invention more specifically
relates to new compounds which are derivatives of isoginkgetin and
are each typically for use as a medicament. In particular, the
invention relates to the use of these new compounds for increasing
the presentation, typically the production and presentation, of
(antigenic) peptides, preferably Pioneer Translation Products
(PTPs)-derived antigens, by cancer cells in a subject, and inducing
or stimulating an immune response in the subject. The immune
response is typically directed against a tumor antigen, more
generally against the cancerous tumour the subject is suffering
of.
[0002] The present disclosure also relates to uses of such
compounds, in particular to prepare a pharmaceutical composition
and/or to allow or improve the efficiency of a cancer therapy in a
subject in need thereof Each of the compounds of the invention can
indeed be advantageously used, in combination with at least one
distinct anticancer agent, typically a chemotherapeutic drug,
and/or with radiotherapy, for treating cancer, for preventing
cancer metastasis and/or for preventing cancer recurrence in a
subject.
[0003] The invention also discloses methods for preventing or
treating cancer, cancer metastasis and/or cancer recurrence in a
subject. The present invention in addition provides kits suitable
for preparing a composition according to the present invention
and/or for implementing the herein described methods.
BACKGROUND OF THE INVENTION
[0004] All nucleated cells present antigenic peptides (APs) at
their surface trough the class I major histocompatibility complex
(MHC-I) pathway. APs are 8 to 10 amino acids long and reflect the
inherent cellular activity (Caron et al.). Because their
presentation guides the surveillance of potentially dangerous
elements by immune cells, mainly cytotoxic CD8.sup.+ T cells (CTL)
and CD4.sup.+ T helper cells, APs are the targets of therapeutic
anti-cancer vaccines currently developed. Despite promising,
clinical trials results with therapeutic vaccines targeting
tumor-associated antigens (TAA) haven't met their expectations. The
main failures have been associated to immunosuppressive mechanisms
and to a suboptimal choice of antigens (Mellman et al.; Burg et
al.). One of the important events that drive tumors
immunoselection, and that is correlated to poor prognosis, is the
loss or the downregulation of MHC class I antigenic presentation by
tumor cells (Watson et al.; Liu et al.). These last can escape CTL
and natural killer cells recognition due to defects in components
of the MHC class I pathway (Leone et al.). Along with the overall
decrease of MHC class I antigenic presentation, the nature of
antigens presented at the cell surface, called the MHCI class I
immunopeptidome (MIP), is of critical importance for immune
recognition. In cancer where a specific TAA is identified and
targeted with immunotherapy such as Her/neu in breast cancer or CEA
in colon cancer, the loss of this TAA expression at the tumor cell
surface leads to immune evasion (Lee et al.; Kmieciak et al.). To
counteract that, current strategies aims at enlarging the range of
targeted cancer peptide and restoring MHC antigenic
presentation.
[0005] In order to understand the dynamic of the MIP, one could
focus on the source of APs for the MHC class I presentation
pathway. Endogenous APs were first thought to strictly come from
the degradation of senescent proteins. However, models suggesting
alternative sources have challenged this notion. In 1996, the group
of J. Yewdell introduces the concept of the Defective ribosomal
products (DRIPs) (Yewdell et al, 1996), initially described as
rapidly degraded products due to their unstable conformation. More
recently, inventors have explored that concept from a different
perspective showing that the major source of APs derive from a
pioneer translation event that occurs before introns are spliced
out and that is independent of the translation event of full length
proteins (Apcher et al., 2011). Produced non-canonical peptides can
therefore be derived from intronic sequence, 3' or 5' UTR regions
as well as alternative reading frames. These polypeptides are
described as Pioneer Translation Products (PTPs). The discovery of
PTPs suggests the existence of a complex translational nuclear
mechanism that partly aims at shaping the MIP by generating
relevant and suitable polypeptides for the MHC class I pathway.
Moreover, PTPs seems to play a role in the dynamic of cancer
development. When inoculated in mouse, it has been shown that
cancer cells presenting PTPs-derived antigens at their surface can
be recognized by specific T-cells leading to tumor growth
reduction. Moreover, purified PTPs containing a model epitope
efficiently promote anti-cancer immune response when injected as a
peptide vaccine in mice (Duvallet et al.).
[0006] Precursor-mRNA (pre-mRNA) splicing is catalyzed in the
nucleus by the spliceosome, a conserved and dynamic multi-protein
complex composed of five small nuclear RNAs (snRNAs) U1, U2, U4, U5
and U6 that are complexes with over 200 proteins. A growing number
of studies report that the deregulation of the spliceosome complex
entails aberrant splicing patters in many cancers contributing to
abnormal tumor cell proliferation and progression. Since 2011,
recurrent spliceosome mutations have been reported in several
cancers, including myelomonocytic leukemia, myeloid leukemia,
chronic lymphocytic leukemia, breast cancers or multiple
myeloma.
[0007] Inventors now herein describe new compounds for use in the
treatment of cancer, in the prevention of cancer metastasis and/or
in the prevention of cancer recurrence in a subject.
SUMMARY OF THE INVENTION
[0008] Inventors produced and herein describe for the first time a
compound of formula
##STR00001##
wherein R.sup.1 and R.sup.2 are independently selected from the
group of Na, H, --CH.sub.3, --CH.sub.2--CH.sub.3,
--CH.sub.2--CH.dbd.CH.sub.3, n-CH.sub.2--CH.sub.2--CH.sub.3,
P(O)(O--CH.sub.2--CH.sub.3).sub.2, P(O)(OH).sub.2 or
P(O)(ONa).sub.2 and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and
R.sup.5 are not H simultaneously, wherein R.sup.1 is not --CH.sub.3
when R.sup.2 is P(O)(ONa).sub.2 or P(O)(OH).sub.2 and each of
R.sup.3, R.sup.4 and R.sup.5 is H, and wherein R.sup.1 is not
--CH.sub.3 or H when R.sup.2 is --CH.sub.3 and each of R.sup.3,
R.sup.4 and R.sup.5 is H; and wherein R.sup.3, R.sup.4 and R.sup.5
are independently selected from the group of H, CH.sub.3,
--CH.sub.2--CH.sub.3, --CH.sub.2--CH.dbd.CH.sub.3, and
C.sub.nH.sub.2n+1 with n=3-10, for use as a medicament.
[0009] This compound, as well as a stereoisomer thereof or as well
as a pharmaceutically acceptable salt thereof, can advantageously
be used as a medicament.
[0010] In a preferred embodiment, the invention relates to a
particular compound of formula (I) wherein R.sup.1 is Na, R.sup.2
is P(O)(ONa).sub.2 and each of R.sup.3, R.sup.4 and R.sup.5 is H
(also herein generally identified as "IP2" or more specifically as
"IP2-6Na"):
##STR00002##
[0011] In another preferred embodiment, the invention relates to a
particular compound of formula (I) wherein R.sup.1 is H, R.sup.2 is
P(O)(ONa).sub.2 and each of R.sup.3, R.sup.4 and R.sup.5 is H (also
herein generally identified as "IP2" or more specifically as
"IP2-4Na"):
##STR00003##
[0012] In a preferred aspect herein described, the compound of
formula (I), preferably a "IP2" compound (IP2-6Na or IP2-4Na), or
stereoisomer or pharmaceutically acceptable salt thereof, is for
use in the treatment of cancer, for use in the prevention of cancer
metastasis and/or for use in the prevention of cancer recurrence in
a subject.
[0013] Further described is the in vivo, in vitro or ex vivo use of
a compound of formula (I), preferably a "IP2" compound (IP2-6Na or
IP2-4Na), for inducing or increasing the presentation, typically
the production and presentation, of (antigenic) peptides,
preferably Pioneer Translation Products (PTPs)-derived antigens, by
cancer cells.
[0014] The compound of formula (I), preferably a "IP2" compound
(IP2-6Na or IP2-4Na), allows the physician to prevent or control,
preferably decrease, cancer cell proliferation by stimulating the
subject's immune system. It is in addition advantageously capable
of increasing the effectiveness of other cancer treatments.
Inventors herein demonstrate that this compound is in addition
capable of reducing the risk of metastasis and/or cancer
recurrence.
[0015] Also herein described is a composition comprising such a
compound of formula (I), preferably a "IP2" compound (IP2-6Na or
IP2-4Na), and a pharmaceutically acceptable carrier, preferably
together with at least one distinct anticancer agent to be used
simultaneously, separately or sequentially. Such a composition is
typically for use for treating cancer, for preventing cancer
metastasis and/or for preventing cancer recurrence in a
subject.
[0016] Also herein described is a method for treating cancer in a
subject, comprising a step of administering a compound, typically
the compound of formula (I), preferably a "IP2" compound (IP2-6Na
or IP2-4Na), or a composition as herein described to the
subject.
[0017] A kit is also described which comprises the compound of
formula (I), preferably a "IP2" compound (IP2-6Na or IP2-4Na), and
preferably at least one distinct anticancer agent in distinct
containers, as well as uses thereof, in particular to prepare a
composition as herein described.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Inventors generated a biflavonoid isoginkgetin derivative
which is described for the first time in the context of the present
invention and is identified as "compound of formula (I)":
##STR00004##
wherein R.sup.1 and R.sup.2 are independently selected from the
group of Na, H, --CH.sub.3, --CH.sub.2--CH.sub.3, --CH.sub.213
CH.dbd.CH.sub.3, n-CH.sub.2--CH.sub.2--CH.sub.3,
P(O)(O--CH.sub.2--CH.sub.3).sub.2, P(O)(OH).sub.2 or
P(O)(ONa).sub.2 and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and
R.sup.5 are not H simultaneously, wherein R.sup.1 is not --CH.sub.3
when R.sup.2 is P(O)(ONa).sub.2 or P(O)(OH).sub.2 and each of
R.sup.3, R.sup.4 and R.sup.5 is H, and wherein R.sup.1 is not
--CH.sub.3 or H when R.sup.2 is --CH.sub.3 and each of R.sup.3,
R.sup.4 and R.sup.5 is H; and wherein R.sup.3, R.sup.4 and R.sup.5
are independently selected from the group of H, CH.sub.3,
--CH.sub.2--CH.sub.3, --CH.sub.2--CH.dbd.CH.sub.3, and
C.sub.nH.sub.2n+1 with n=3-10, preferably n=3-8.
[0019] A "hydroxymethyl" refers to a radical of formula --OMe
wherein Me represents a methyl (--CH.sub.3).
[0020] A "sodium hydroxide" refers to a radical of formula --ONa
wherein Na represents a sodium.
[0021] The C.sub.nH.sub.2n+1 group where n=3-10 refers to an
"alkyl" group which is a saturated, linear or branched aliphatic
group. It includes for instance propyl, isopropyl, butyl, isobutyl,
tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl group.
Preferably, n is from 3-8 or from 3-6.
[0022] A particular compound of formula (I) wherein R.sup.1 is Na,
R.sup.2 is P(O)(ONa).sub.2 and each of R.sup.3, R.sup.4 and R.sup.5
is H is also herein generally identified as "IP2" molecule or more
specifically as "IP2-6Na", or as Sodium
8-(2-methoxy-5-(5-oxido-4-oxo-7-(phosphonatooxy)-4H-chromen-2-yl)phenyl)--
2-(4-methoxyphenyl)-5-oxido-4-oxo-4H-chromen-7-yl phosphate:
##STR00005##
[0023] Another particular compound of formula (I) wherein R.sup.1
is H, R.sup.2 is P(O)(ONa).sub.2 and each of R.sup.3, R.sup.4 and
R.sup.5 is H is also herein generally identified as "IP2" molecule
or more specifically as "IP2-4Na" or as sodium
5-hydroxy-8-(5-(5-hydroxy-4-oxo-7-(phosphonatooxy)-4H-chromen-2-yl)-2-met-
hoxyphenyl)-2-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl
phosphate:
##STR00006##
[0024] The compound of formula (I), preferably a "IP2" compound
(IP2-6Na or IP2-4Na), as well as a stereoisomer thereof or a
pharmaceutically acceptable salt thereof, can advantageously be
used as a medicament.
[0025] As used herein, the term "pharmaceutically acceptable"
refers to compositions, compounds, salts and the like that are,
within the scope of sound medical judgment, suitable for contact
with the tissues of the subject, or which can be administered to
the subject, without excessive toxicity or other complications
commensurate with a reasonable benefit/risk ratio. For instance,
pharmaceutically acceptable salts encompass sodium, potassium,
chloride, ammonium, acetate salts and the like.
[0026] Inventors herein demonstrate that the compound of formula
(I), preferably a "IP2" compound (IP2-6Na or IP2-4Na), can
advantageously be used as a positive immunomodulator against
cancer.
[0027] Inventors looked at the antigenic presentation of a
PTPs-derived antigen model expressed in human and mouse cancer cell
lines and observed that their in vitro treatment with isoginkgetin
and, more preferably IP2, increases the presentation of this
antigen. In addition, they showed that in vivo treatment with the
isoginkgetin dissolved in DMSO of sarcoma-bearing mice slows down
tumor growth in an immune-dependent manner. In order to ameliorate
its effect, they tested the isoginkgetin derivative IP2 that is
soluble in water and surprisingly observed that it is a far more
potent inhibitor of cancer growth than isoginkgetin itself Since in
immunodeficient Nu/Nu mice, the natural product and the derivative
have no effect on tumor growth they concluded that their effects
are dependent on the immune response. Those results demonstrate
that PTPs-derived antigenic presentation can be modulated and
inventors provide a new promising molecule for market development:
the IP2 splicing inhibitor or compound of formula (I) which can be
used to boost the anti-cancer response and treat cancer contrary to
other derivatives of isoginkgetin.
[0028] In a preferred aspect herein described, the compound of
formula (I), preferably a "IP2" compound (IP2-6Na or IP2-4Na), is
for use in the treatment of cancer, for use in the prevention of
cancer metastasis and/or for use in the prevention of cancer
recurrence in a subject.
[0029] In another preferred aspect herein described, the compound
of formula (I), preferably a "IP2" compound (IP2-6Na or IP2-4Na),
is for use for stimulating an anti-cancer immune response in a
subject in need thereof.
[0030] In a further preferred aspect, the compound of formula (I),
preferably a "IP2" compound (IP2-6Na or IP2-4Na), is for use for
inducing or increasing the presentation, typically the production
and presentation, of Pioneer Translation Products (PTPs)-derived
antigens by cancer cells.
[0031] The compound of the invention can be obtained by methods
well-known by the skilled artisan such as hemi-synthesis or total
synthesis. An example of a method for producing the compound of
formula (I) is herein described in the experimental part and
further illustrated on FIG. 5 (the compound of formula (I)
corresponds to compound "2" and "2") on FIG. 5, which is generally
herein identified as "IP2"). The compound of formula (I) is an
artificial product which cannot be found as such in nature.
[0032] The compound of formula (I) can be typically prepared from
the biflavonoid Isoginkgetin which has been described as a general
inhibitor of mRNA splicing and is typically extracted from leaves
of maidenhair tree, Ginko biloba L. Methods for extracting
Isoginkgetin are described, among others, in Kang et al (1990) and
in Lee et al (1995), the disclosure of which being incorporated
herein by reference.
[0033] The compound of formula (I) can also be prepared by chemical
synthesis by using conventional chemical reactions.
[0034] A further object of the invention is the use of a compound
of formula (I), preferably a "IP2" compound (IP2-6Na or IP2-4Na),
(or a stereoisomer or a pharmaceutically acceptable salt thereof)
for decreasing the resistance of a cancer or subject suffering of
cancer with respect to a distinct anticancer agent, typically a
distinct chemotherapeutic agent.
[0035] Also herein described is a compound of formula (I),
preferably a "IP2" compound (IP2-6Na or IP2-4Na), according to the
invention (or a stereoisomer or a pharmaceutically acceptable salt
thereof), or a composition comprising such a compound and a
pharmaceutically acceptable carrier, for use, in combination with
at least one distinct anticancer agent, typically a distinct
chemotherapeutic drug, and/or with radiotherapy, for treating
cancer, for preventing cancer metastasis and/or for preventing
cancer recurrence in a subject.
[0036] The term "subject" refers to any subject, preferably a
mammal
[0037] Examples of mammals include humans and non-human animals
such as, without limitation, domesticated animals (e.g., cows,
sheep, cats, dogs, and horses), non-human primates (such as
monkeys), rabbits, and rodents (e.g., mice and rats).The treatment
is preferably intended for a human being in need thereof, whatever
its age or sex.
[0038] The term "subject" typically designates a patient, in
particular a patient having a tumor. Unless otherwise specified in
the present disclosure, the tumor is a cancerous or malignant
tumor. In a particular aspect, the subject is a subject undergoing
a treatment of cancer such as chemotherapy and/or radiotherapy, or
a subject at risk, or suspected to be at risk, of developing a
cancer.
[0039] The subject is, for example a human being suffering of a
cancer and resistant to cancer treatment, typically to
chemotherapy.
[0040] The subject may have been exposed to part of a complete
conventional treatment protocol, for example to at least one cycle
of the all treatment protocol, for example two cycles of the all
treatment protocol.
[0041] The cancer or tumor may be any kind of cancer or neoplasia.
The tumor is typically a solid tumor, in particular of epithelial,
neuroectodermal or mesenchymal origin. The cancer is also typically
selected from a carcinoma, sarcoma, lymphoma, germ cell tumor,
blastoma, leukemia and multiple myeloma, preferably from a
carcinoma, sarcoma, blastoma, lymphoma, leukemia and multiple
myeloma. The cancer can be a metastatic cancer or not.
[0042] The cancer can for example be selected from, without being
limited to, the group consisting of chronic myeloid leukemia, acute
lymphoblastic leukemia, Philadelphia chromosome positive acute
lymphoblastic leukemia (Ph.sup.+ ALL), Hodgkin's disease, Hodgkin's
or non-Hodgkin lymphoma, squamous cell carcinoma, small-cell lung
cancer, non-small cell lung cancer, glioma, gastrointestinal
cancer, renal cancer, ovarian cancer, liver cancer, colorectal
cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid
cancer, neuroblastoma, brain cancer, central nervous system cancer,
pancreatic cancer, glioblastoma multiforme, cervical cancer,
stomach cancer, bladder cancer, malignant hepatoma, breast cancer,
colon carcinoma, head and neck cancer, gastric cancer, germ cell
tumor, pediatric sarcoma, rhabdomyosarcoma, Ewing's sarcoma,
osteosarcoma, soft tissue sarcoma, sinonasal NK/T-cell lymphoma,
myeloma, melanoma, multiple myeloma, acute myelogenous leukemia
(AML), and chronic lymphocytic leukemia.
[0043] In a preferred embodiment, the cancer is selected from the
group consisting of lung cancer, breast cancer, genito-urinary
cancer (such as prostate cancer, bladder cancer, testis cancer,
uterine cervix cancer or ovaries cancer) and sarcoma (such as
osteosarcoma or soft tissue sarcoma, including pediatric soft
tissue sarcoma, neuroblastoma, myeloma and melanoma).
[0044] More preferably, the cancer is selected from melanoma, lung
cancer (including non-small-cell lung carcinoma (or NSCLC) and
small-cell lung carcinoma (or SCLC)) and breast cancer.
[0045] Even more preferably, the carcinoma is a melanoma or a lung
cancer.
[0046] In an aspect, the cancer is a lung cancer, typically a
small-cell lung cancer or a non-small cell lung cancer.
[0047] In another aspect, the cancer is a leukemia, typically an
acute myelogenous leukemia (AML) or a chronic lymphocytic
leukemia.
[0048] In a further aspect, the cancer is a colon cancer, typically
a colon carcinoma. The cancer may also be a colorectal cancer.
[0049] In a further aspect, the cancer is a pediatric cancer
typically a pediatric sarcoma, lymphoma, leukemia, neuroblastoma,
brain cancer, or central nervous system cancer.
[0050] In a particular aspect herein described, the anticancer
agent is selected from a chemotherapeutic agent, an immune
checkpoint blocker and an anti-cancer vaccine (also herein
identified as "cancer vaccine"). These agents are typically
considered as "conventional" agents for treating cancer.
[0051] The chemotherapeutic agent is typically an agent selected
for example from an antitumor/cytotoxic antibiotic, an alkylating
agent, an antimetabolite, a topoisomerase inhibitor, a mitotic
inhibitor, a platin based component, a specific kinase inhibitor,
an hormone, a cytokine, an antiangiogenic agent, an antibody, a DNA
methyltransferase inhibitor and a vascular disrupting agent.
[0052] The antitumor agent or cytotoxic antibiotic can for example
be selected from an anthracycline (e.g. doxorubicin, daunorubicin,
adriamycine, idarubicin, epirubicin, mitoxantrone, valrubicin),
actinomycin, bleomycin, mitomycin C, plicamycin and
hydroxyurea.
[0053] The alkylating agent can for example be selected from
mechlorethamine, cyclophosphamide, melphalan, chlorambucil,
ifosfamide, temozolomide busulfan, N-Nitroso-N-methylurea (MNU),
carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU),
fotemustine, streptozotocin, dacarbazine, mitozolomide, thiotepa,
mytomycin, diaziquone (AZQ), procarbazine, hexamethylmelamine and
uramustine
[0054] The antimetabolite can for example be selected from a
pyrimidine analogue (e.g. a fluoropyrimidine analog, 5-fluorouracil
(5-FU), floxuridine (FUDR), cytosine arabinoside (Cytarabine),
Gemcitabine (Gemzar.RTM.), capecitabine); a purine analogue (e.g.
azathioprine, mercaptopurine, thioguanine, fludarabine,
pentostatin, cladribine, clofarabine); a folic acid analogue (e.g.
methotrexate, folic acid, pemetrexed, aminopterin, raltitrexed,
trimethoprim, pyrimethamine).
[0055] The topoisomerase inhibitor can for example be selected from
camptothecin, irinotecan, topotecan, amsacrine, etoposide,
etoposide phosphate and teniposide.
[0056] The mitotic inhibitor can for example be selected from a
taxane [paclitaxel (PG-paclitaxel and DHA-paclitaxel) (Taxol.RTM.),
docetaxel (Taxotere.RTM.), larotaxel, cabazitaxel, ortataxel,
tesetaxel, or taxoprexin]; a spindle poison or a vinca alkaloid
(e.g. vincristine, vinblastine, vinorelbine, vindesine or
vinflunine); mebendazole; and colchicine.
[0057] The platin based component can for example be selected from
platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin,
satraplatin and triplatin tetranitrate.
[0058] The specific kinase inhibitor can for example be selected
from a BRAF kinase inhibitor such as vemurafenib; a MAPK inhibitor
(such as dabrafenib); a MEK inhibitor (such as trametinib); and a
tyrosine kinase inhibitor such as imatinib, gefitinib, erlotinib,
sunitinib or carbozantinib.
[0059] Tamoxifen, an anti-aromatase, or an anti-estrogen drug can
also typically be used in the context of hormonotherapy.
[0060] A cytokine usable in the context of an immunotherapy can be
selected for example from IL-2 (Interleukine-2), IL-11
(Interleukine-11), IFN (Interferon) alpha (IFNa), and
Granulocyte-macrophage colony-stimulating factor (GM-CSF).
[0061] The anti-angiogenic agent can be selected for example from
bevacizumab, sorafenib, sunitinib, pazopanib and everolimus.
[0062] The antibody, in particular the monoclonal antibody (mAb)
can be selected from a anti-CD20 antibody (anti-pan B-Cell
antigen), anti-Her2/Neu (Human Epidermal Growth Factor
Receptor-2/NEU) antibody; an antibody targeting cancer cell surface
(such as rituximab and alemtuzumab); a antibody targeting growth
factor (such as bevacizumab, cetuximab, panitumumab and
trastuzumab); a agonistic antibody (such as anti-ICOS mAb,
anti-OX40 mAb, anti-41BB mAb); and an immunoconjugate (such as
90Y-ibritumomab tiuxetan, 131I-tositumomab, or ado-trastuzumab
emtansine).
[0063] A DNA methyltransferase inhibitor can for example be
selected from 2'-deoxy-5-azacytidine (DAC), 5-azacytidine,
5-aza-2'-deoxycytidine, 1-[beta]-D-arabinofuranosyl-5-azacytosine
and dihydro-5-azacytidine.
[0064] A vascular disrupting agent can for example be selected from
a flavone acetic acid derivative, 5,6-dimethylxanthenone-4-acetic
acid (DMXAA) and flavone acetic acid (FAA).
[0065] Other chemotherapeutic drugs include a proteasome inhibitor
(such as bortezomib), a DNA strand break compound (such as
tirapazamine), an inhibitor of both thioredoxin reductase and
ribonucleotide reductase (such as xcytrin), and an enhancer of the
Thl immune response (such as thymalfasin).
[0066] In a preferred embodiment, the chemotherapeutic drug or
agent is selected from an antitumor/cytotoxic antibiotic, an
alkylating agent, an antimetabolite, a topoisomerase inhibitor, a
mitotic inhibitor, a platin based component, a specific kinase
inhibitor, an antiangiogenic agent, an antibody and a DNA
methyltransferase inhibitor.
[0067] An immune checkpoint blocker is typically an antibody
targeting an immune checkpoint. Such an immune checkpoint blocker
can be advantageously selected from anti-CTLA4 (ipilimumab and
Tremelimumab), anti-PD-1 (Nivolumab and Pembrolizumab), anti-PD-L1
(Atezolizumab, Durvalumab, and Avelumab), anti-PD-L2 and
anti-Tim3.
[0068] The cancer vaccine can for example be selected from a
vaccine composition comprising (antigenic) peptides, in particular
PTPs; a Human papillomavirus (HPV) vaccine (such as Gardasil.RTM.,
Gardasil9.RTM., and Cervarix.RTM.); a vaccine stimulating an immune
response to prostatic acid phosphatase (PAP) sipuleucel-T
(Provenge.RTM.); an oncolytic virus; and talimogene laherparepvec
(T-VEC or Imlygic.RTM.).
[0069] In another particular aspect, the ("conventional") cancer
treatment is an irradiation (also herein identified as
"radiotherapy"). The radiotherapy typically involves rays selected
from X-rays ("XR"), gamma rays and/or UVC rays.
[0070] The treatment which can include several anticancer agents is
selected by the cancerologist depending on the specific cancer to
be prevented or treated.
[0071] A particular melanoma is a melanoma conventionally treated
with ipilimumab, nivolumab, pembrolizumab, IFNa, dacarbazine, a
BRAF inhibitor, dabrafenib, trametinib, sorafenib, temozolomide,
electrochemotherapy, TNFalpha and/or fotemustine.
[0072] In a particular embodiment, the melanoma is a melanoma
resistant to the previously described cytotoxic conventional
therapies.
[0073] A particular breast cancer is a breast cancer conventionally
treated with an anthracycline, a taxane, trastuzumab, an anti-PARP
(Poly (ADP-ribose) polymerase), an anti-PI3K (Phosphoinositide
3-kinase), a mTOR (mammalian Target of Rapamycin) inhibitor,
vinorelbine, gemcitabine, an antioestrogen, and/or an
antiaromatase, before or after a surgical step to remove breast
tumor, preferably before such a surgical step.
[0074] In a particular embodiment, the breast cancer is a breast
cancer resistant to the previously described conventional
therapies.
[0075] A particular lung cancer is a lung cancer conventionally
treated with XR and either platine or permetrexed.
[0076] A particular early stage NSCLC is an NSCLC conventionally
treated with paclitaxel, docetaxel gemcitabine, vinorelbine,
etoposide, taxane, avastin [anti-VEGF (Vascular endothelial growth
factor) antibody], erlotinib and/or gefitinib. In a particular
embodiment, the lung cancer is resistant to conventional
therapies.
[0077] The present disclosure further relates to the use of the
compound of formula (I) of the invention, preferably "IP2", to
prepare a pharmaceutical composition or medicament, said
composition being capable of treating cancer or of improving the
efficiency of a therapy of cancer in a subject in need thereof by
stimulating the subject's immune system. The compound of the
invention can in particular be advantageously used, in combination
with at least one distinct anti-cancer agent as described
previously or any other therapeutically active compound, and/or
with radiotherapy, for treating cancer, for preventing cancer
metastasis and/or for preventing cancer recurrence in a
subject.
[0078] Also herein described is thus a composition comprising,
typically as a combined preparation, a compound of formula (I),
preferably a "IP2" compound (IP2-6Na or IP2-4Na), and a
pharmaceutically acceptable carrier, preferably together with at
least one distinct anticancer agent, for simultaneous, separate or
sequential use in the treatment of said cancer.
[0079] Herein described are also (i) a method for preventing or
treating cancer, (ii) a method for increasing the sensitivity of a
cancer to an anticancer agent, and (iii) a method for decreasing
the resistance of a cancer with respect to an anticancer agent,
each of said methods comprising administering a subject in need
thereof with an effective amount, typically a therapeutically
effective amount, of at least one compound of formula (I),
preferably a "IP2" compound (IP2-6Na or IP2-4Na), or a
pharmaceutical composition as defined above, preferably together
with an anticancer agent classically used in the prevention or
treatment of cancer as herein described (as a combined
preparation).
[0080] In another particular aspect, said method further comprises
administering an effective amount of another therapeutically active
compound for preventing or treating cancer or a cancer treatment
side effect.
[0081] As used herein, "treatment" or "treat" refers to therapeutic
intervention in an attempt to alter the natural course of the
subject being treated, and can be performed either for preventive
(prophylactic) or curative purpose. Desirable effects of treatment
include, but are not limited to, preventing occurrence or
recurrence of disease, alleviation of symptoms, and diminishment of
any direct or indirect pathological consequences of the disease,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis. In preferred embodiments, compositions and methods of
the invention are used to delay development of a cancer or to slow
the progression of a cancer, typically of tumor growth.
[0082] Typically, the treatment will induce a therapeutic response
of the immune system of the subject, typically CD4.sup.+ and/or
CD8.sup.+ T cells response(s).
[0083] By inducing a T cell response is typically meant herein that
a T cell response directed towards a certain antigen is elicited.
Before said induction, said T cell response was not present, or
below detection levels or not functional. By enhancing a T cell
response is meant herein that the overall action of T cells
directed towards a certain antigen is made higher and/or more
efficient compared to the overall action of said T cells before
said enhancement. For instance, after said enhancement more T cells
directed towards said antigen may be generated. As a result, the
action of the additionally generated T cells increases the overall
action against said antigen. Alternatively, said enhancement may
comprise the increment of the action of T cells directed towards
said antigen. Said T cells may for instance react stronger and/or
quicker with said antigen. Of course, the result of said
enhancement may be generation of additional T cells together with
increment of the action of said T cells. Alternatively, said
enhancement may comprise generation of additional T cells, or
increment of the action of T cells, only.
[0084] Another object herein described relates to a method of
producing an immune response in a subject, typically against a
specific target, preferably a tumor antigen or cancer/tumor cell or
tissue, the method comprising injecting to said subject a compound
of formula (I) according to the invention or composition according
to the invention comprising such a compound, typically in an
effective amount. The detection of a therapeutic immune response
can be easily determined by the skilled person thanks to
technologies such as ELISA, ELISPOT, delayed type hypersensitivity
response, intracellular cytokine staining, and/or extracellular
cytokine staining.
[0085] As used herein, "an effective amount or dose" or "a
therapeutically effective amount or dose" refers to an amount of
the compound of the invention which prevents, removes, slows down
the cancer or reduces or delays one or several symptoms or
disorders caused by or associated with said disease in the subject,
or which induce a measurable immune response in the subject, who is
preferably a human being. The effective amount, and more generally
the dosage regimen, of the compound of the invention and
pharmaceutical compositions thereof may be determined and adapted
by the one skilled in the art. An effective dose can be determined
by the use of conventional techniques and by observing results
obtained under analogous circumstances. The therapeutically
effective dose of the compound of the invention will vary depending
on the disease to be treated or prevented, its gravity, the route
of administration, any co-therapy involved, the patient's age,
weight, general medical condition, medical history, etc.
[0086] Typically, the amount of the compound to be administrated to
a patient may range from about 0.01 mg/kg to 500 mg/kg of body
weight for a human patient. In a particular embodiment, the
pharmaceutical composition according to the invention comprises 0.1
mg/kg to 100 mg/kg of the compound of the invention, for instance
from 0.5 mg/kg to 10 mg/kg.
[0087] In a particular aspect, the compounds of the invention can
be administered to the subject by parenteral route, oral route, or
intraveinous (IV), intratumoral (IT) or intraperitoneal (IP)
injection. The compound or the nanoparticle of the invention may be
administered to the subject daily (1time a day) during several
consecutive days, for example during 2 to 10 consecutive days,
preferably from 3 to 6 consecutive days. Said treatment may be
repeated during 1, 2, 3, 4, 5, 6 or 7 weeks, or every two or three
weeks or every one, two or three months. Alternatively, several
treatment cycles can be performed, optionally with a break period
between two treatment cycles, for instance of 1, 2, 3, 4 or 5
weeks. The compound of the invention can for example be
administered as a single dose once a week, once every two weeks, or
once a month. The treatment may be repeated one or several times
per year. Doses are administered at appropriate intervals which can
be determined by the skilled person. The amount chosen will depend
on multiple factors, including the route of administration,
duration of administration, time of administration, the elimination
rate of the selected compound of formula (I), or of the various
products used in combination with said compound, the age, weight
and physical condition of the patient and his/her medical history,
and any other information known in medicine.
[0088] The administration route can be performed by various routes.
For example it can be oral or parenteral. It is typically performed
by systemic injection, e.g., intravenous, intra-muscular,
intra-peritoneal, intra-tumoral, sub-cutaneous, etc. The
pharmaceutical composition is adapted for one or several of the
above-mentioned routes. The pharmaceutical composition is
preferably administered by injection or by intravenous infusion of
suitable sterile solutions, or in the form of liquid or solid doses
via the alimentary canal.
[0089] The pharmaceutical composition can be formulated as
solutions in pharmaceutically compatible solvents or vehicles, or
as pills, tablets, capsules, powders, suppositories, etc. that
contain solid vehicles in a way known in the art, possibly through
dosage forms or devices providing sustained and/or delayed release.
For this type of formulation, an agent such as cellulose, lipids,
carbonates or starches are used advantageously.
[0090] Agents or vehicles that can be used in the formulations
(liquid and/or injectable and/or solid) are excipients or inert
vehicles, i.e. pharmaceutically inactive and non-toxic
vehicles.
[0091] Mention may be made, for example, of saline, physiological,
isotonic and/or buffered solutions, compatible with pharmaceutical
use and known to those skilled in the art. The compositions may
contain one or more agents or vehicles chosen from dispersants,
solubilizers, stabilizers, preservatives, etc.
[0092] Formulations of the present invention suitable for oral
administration may be in the form of discrete units as capsules,
sachets, tablets or lozenges, each containing a predetermined
amount of the active ingredient; in the form of a powder or
granules; in the form of a solution or a suspension in an aqueous
liquid or non-aqueous liquid; or in the form of an oil-in-water
emulsion or a water-in-oil emulsion. Formulations suitable for
parenteral administration conveniently comprise a sterile oily or
aqueous preparation of the active ingredient which is preferably
isotonic with the blood of the recipient. Every such formulation
can also contain other pharmaceutically compatible and non-toxic
auxiliary agents, such as, e.g. stabilizers, antioxidants, binders,
dyes, emulsifiers or flavouring substances.
[0093] The formulations of the present invention comprise an active
ingredient, the compound of formula (I), preferably "IP2", in
association with a pharmaceutically acceptable carrier and
optionally with other active or therapeutic ingredients. The
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of the formulations and not deleterious to
the recipient thereof Methods for the safe and effective
administration of most of these anti-cancer agents are known to
those skilled in the art. In addition, their administration is
described in the standard literature.
[0094] Another object of the invention is a kit comprising at least
one compound of formula (I) according to the invention, preferably
"IP2", and preferably at least one distinct anticancer agent,
typically chemotherapeutic drug, in distinct containers. The kit
can further comprise instructions for preparing a composition
according to the invention, for carrying out anyone of the herein
described method, for example for preventing or treating cancer,
for preventing or treating cancer metastasis and/or for preventing
or treating cancer recurrence in a subject.
[0095] In a particular embodiment, the present invention relates to
the use of a kit according to the invention to prepare a
composition as herein described.
[0096] In another particular embodiment, the kit is suitable for
implementing anyone of the herein described method, in particular a
method for treating cancer, for preventing cancer metastasis and/or
for preventing cancer recurrence in a subject.
[0097] Further aspects and advantages of the present invention will
be disclosed in the following experimental section and figures
which shall be considered as illustrative only.
LEGENDS TO THE FIGURES
[0098] FIG. 1: Isoginkgetin treatment increases antigenic
presentation of intron-derived antigens in cancer cells.
[0099] B3Z specific T-cell activation after treatment with 2,5
.mu.M and 6,25 .mu.M isoginkgetin of (A) human melanoma cell line
A375, (B) human lung carcinoma cell line A549, (C) normal lung
fibroblast cell line MRC5. (D) B3Z specific T-cell activation after
treatment with 6,25 .mu.M and 15 .mu.M isoginkgetin of mouse
melanoma cell line B16F10. (E) B3Z specific T-cell activation after
treatment with 15 .mu.M and 25 .mu.M isoginkgetin of mouse sarcoma
cell line MCA205.
[0100] FIG. 2: Isoginkgetin treatment increases antigenic
presentation of intron-derived antigens in cancer cells.
[0101] B3Z specific T-cell activation after treatment with 6,25
.mu.M, 15 .mu.M and 25 .mu.M isoginkgetin of MCA205 cells
previously transfected with (A) globin-SL8-intron construct, (B)
globin-SL8-exon construct, (C) Ovalbumin construct. B3Z specific
T-cell activation after treatment with 6,25 .mu.M, 15 .mu.M and 25
.mu.M isoginkgetin of B16F10 cells previously transfected with (D)
globin-SL8-intron construct, (E) globin-SL8-exon construct, (F)
Ovalbumin construct. Data are given as mean .+-.SEM. *P<0.05,
**P<0.01, ***P<0.001 (unpaired student t test).
[0102] FIG. 3: Isoginkgetin treatment slows clown tumor growth in
vivo.
[0103] (A) Experimental settings. (B) MCA205 globin-SL8-intron
cells were inoculated subcutaneously on the mice flank. Five days
and fifteen days later, 6 mg/kg, 12 mg/kg or 18 mg/kg isoginkgetin
was injected intraperitoneally. Tumor size was assessed every 3 to
4 days until day 27. Each line represents the tumor size in area
(mm.sup.2) of 6 mice in each group. Data are given as mean .+-.SEM.
*p<0.05 (ANOVA with Tukey's multiple comparison test comparing
all groups).
[0104] FIG. 4: Isoginkgetin derivative IP2 efficiently increases
MHC class I presentation of intron-derived antigen in vitro and
reduces tumor growth in vivo in an immune-dependent manner.
[0105] B3Z specific T-cell activation after treatment of MCA205
cells expressing intron-derived SL8 antigen with 15 .mu.M, 25 .mu.M
and 35 .mu.M (A) IP2 or (B) IM2P2, (C) product 10. (D) MCA205
globin-SL8-intron cells were inoculated subcutaneously on the
C56BL/7 mice flank. Five days and fifteen days later, PBS or 18
mg/kg of isoginkgetin, IP2, or M2P2 was injected intraperitoneally.
Tumor size was assessed every 3 to 4 days until day 27. Each line
represents the tumor size in area (mm.sup.2) of 6 mice in each
group. Data are given as mean .+-.SEM. *p<0.05, **p<0.01
(ANOVA with Tukey's multiple comparaison test comparing all
groups). (E) MCA205 globin-SL8-intron cells were inoculated
subcutaneously on the Nude nu/nu mice flank. Five days and fifteen
days later, PBS, 18 mg/kg of isoginkgetin or IP2 was injected
intraperitoneally. Tumor size was assessed every 3 to 4 days until
day 27. Each line represents the tumor size in area (mm.sup.2) of 6
mice in each group. Data are given as mean .+-.SEM.
[0106] FIG. 5: Schema of synthesis of isoginkgetin derivatives.
[0107] FIG. 6: Schema of synthesis of isoginkgetin derivatives.
[0108] FIG. 7: Splicing inhibition increases exon- and
intron-derived antigens MHC-I presentation in cancer cells.
[0109] B3Z SL8-specific T-cell activation after co-culture with (A)
human melanoma A375, human lung carcinoma A549 or normal human lung
fibroblast MRC5 cell lines, all transiently expressing the
intron-derived SL8 peptide and the H2-K.sup.b molecules and treated
upstream with 2,5 .mu.M or 6,25 .mu.M isoginkgetin for 18 hours; or
with (B) mouse sarcoma MCA205 or mouse melanoma B16F10 cell lines
both transiently expressing the intron-derived SL8 peptide and
treated upstream with 6,25 .mu.M, 15 .mu.M or 25 .mu.M isoginkgetin
for 18 hours. B3Z activation after co-culture with MCA205 or B16F10
cells that both transiently express (C) the exon-derived SL8
peptides or (D) the Ova cDNA construct, which doesn't need to be
spliced, treated upstream with 6,25 .mu.M, 15 .mu.M or 25 .mu.M
isoginkgetin for 18 hours. Free SL8 peptides were added in each
condition to ensure that T-cell assays were carried out at
non-saturated conditions and that the expression of MHC-I molecules
was taking into account in the results. Each graph is one
representative of at least four independent experiments.
[0110] Data are given as mean .+-.SEM. *P<0.05, **P<0.01,
***P<0.001 (unpaired student t test).
[0111] FIG. 8: Expression of H2-K.sup.b molecules at the cells
surface.
[0112] Flow cytometry analyses of H2-K.sup.b expression on MCA205
and B16F10 cells treated with (A) isoginkgetin. Flow cytometry
analyses of transiently expressed H2-K.sup.b expression on A375,
A549 and MRC5 cells treated with (B) isoginkgetin.
[0113] FIG. 9: Isoginkgetin splicing inhibitor reduces the growth
of tumor bearing intron-derived-SL8 in an immune-dependent
manner.
[0114] (A) Experimental settings. Growth of (B) sarcoma MCA205 or
(C) melanoma B16F10 cells that both stably express the
globin-SL8-intron construct or (D) MCA205 Wild Type (WT) or (E)
B16F10 WT cells that were subcutaneously inoculated on the flank of
immunocompetent C57BL/6 mice injected intraperitoneally with 12
mg/kg or 18 mg/kg of isoginkgetin at days 5, 10 and 15 after
inoculation. Tumor size was assessed every 3 to 4 days until
reaching the established ethical endpoints. Each line represents
the tumor size in area (mm.sup.2) of 6 mice in each group. Size in
area (mm.sup.2) of (F) MCA205 globin-SL8-intron, (G) B16F10
globin-SL8-intron, (H) MCA205 WT or (I) B16F10 WT tumors
subcutaneously inoculated on the flank of immunodeficient Nu/Nu
Nude mice injected intraperitoneally with 18 mg/kg of isoginkgetin
at days 5, 10 and 15 after inoculation. Data are presented at the
day before the endpoints are reached.
[0115] Data are given as mean .+-.SEM. *p<0.05, **p<0.01
(ANOVA with Tukey's multiple comparison test comparing all
groups).
[0116] FIG. 10: Synthesis and activity of the isoginkgetin
derivatives IP2 and M2P2 (also herein identified as "IM2P2").
[0117] Molecular structure of (A) isoginkgetin, (B) IP2 (IP2-6Na
and IP2-4Na) and (C) M2P2 compounds. (D) B16F10 globin-SL8-intron
or (E) MCA205 globin-SL8-intron were treated with 15 .mu.M
isoginkgetin, 35 .mu.M IP2 or 35 .mu.M M2P2 for 48 hours. RNA was
extracted and qRT-PCR was performed with primers amplifying the
unspliced (intron) and the spliced (exon) globin-SL8-intron RNA.
Data are given as mean .+-.SEM of the ratio of
2.sup.-.DELTA..DELTA.Ct intron and 2.sup.-.DELTA..DELTA.Ct exon of
at least three independent experiments. MTT assay performed on
MCA205 or B16F10 cells treated with 15 .mu.M or 35 .mu.M of (F) IP2
or (G) M2P2. Data are express as mean .+-.SEM of the percentage of
viable cells compared to the control condition of at least three
independent experiments. *P<0.05, **P<0.01, ***P<0.001
(unpaired student t test).
[0118] FIG. 11: IP2 treatment reduces tumor growth and extends
survival.
[0119] B3Z SL8-specific T-cell activation after co-culture with
mouse (A) sarcoma MCA205 or (B) melanoma B16F10 cell lines both
transiently expressing the intron-derived SL8 peptide and treated
upstream with 15 .mu.M or 35 .mu.M of IP2 (left panels) or of M2P2
(right panel). Data are given as mean .+-.SEM. *P<0.05,
**P<0.01, ***P<0.001 (unpaired student t test). Growth of (C)
MCA205 (left panel) or melanoma B16F10 cells (right panel) that
both stably express the globin-SL8-intron construct or (D) MCA205
WT (left panel) or B16F10 WT (right panel) cells that were
subcutaneously inoculated on the flank of immunocompetent C57BL/6
mice injected intraperitoneally with 18 mg/kg of isoginkgetin, of
M2P2 or of IP2 or 24 mg/kg or 36 mg/kg of IP2 at day 5, 10 and 15
after inoculation. Tumor size was assessed every 3 to 4 days until
reaching the established ethical endpoints. Each line represents
the tumor size in area (mm.sup.2) of at least 6 mice in each group.
Data are given as mean .+-.SEM. *p<0.05, **p<0.01 (ANOVA with
Tukey's multiple comparaison test comparing all groups). Kaplan
Meier plots of (E) MCA205 globin-SL8-intron cells inoculated
subcutaneously on the flank of immunocompetent C57BL/6 mice
injected intraperitoneally with PBS or 18 mg/kg of isoginkgetin, of
M2P2 or of IP2. A Log-rank (Mantel-Cox) test was performed.
[0120] FIG. 12: IP2 does not impact H2-K.sup.b molecules expression
at the cell surface and does not induce apoptosis
[0121] Flow cytometry analyses of H2-K.sup.b expression on (A)
MCA205 and (B) B16F10 cells treated with IP2 and M2P2. (C) Flow
cytometry analyses of early, late and total apoptotic MCA205 and
B16F10 cells treated with 35 .mu.M or 1000 .mu.M IP2 for 18
hours.
[0122] FIG. 13: IP2 therapeutic effect is dependent on the immune
response
[0123] Growth curve of (A) MCA205 globin-SL8-intron (left panel),
MCA205 WT (right panel), (B) Bl6F10 globin-SL8-intron (left panel)
or B16F10 WT (right panel) subcutaneously inoculated on the flank
of immunodeficient Nu/Nu Nude mice, intraperitoneally injected at
days 5, 10 and 15 with 18, 24 or 36 mg/kg of IP2. Growth curve of
(C, left panel) MCA205 globin-SL8-intron subcutaneously inoculated
on the flank of immunocompetent mice treated with PBS or 24 mg/kg
of IP2 at day 5, 10 and 15 after inoculation as well as with in
vivo anti-CD8 or isotype every 3 days. Each line represents the
tumor size in area (mm.sup.2) of at least 6 mice in each group. The
C right panel represents the tumor size at day 27. Data are given
as mean .+-.SEM. *p<0.05, **p<0.01 (ANOVA with Tukey's
multiple comparison test comparing all groups). (D) Growth curve of
MCA205 globin-SL8-intron cells or B16F10 WT cells inoculated in 100
days tumor free C57BL/6 mice previously inoculated with MCA205
globin-SL8-intron and treated with IP2 (left panel) or isoginkgetin
(right panel). Each line represents the tumor size in area
(mm.sup.2) of at least 4 mice in each group.
[0124] FIG. 14: IP2 treatment reduces tumor growth from established
tumor
[0125] Growth of sarcoma 15*10.sup.5 MCA205 cells that stably
express the globin-SL8-intron construct were subcutaneously
inoculated on the flank of immunocompetent C57BL/6 mice. Tumors
were allowed to progress 10 days before being ranked and assigned
to groups of equivalent tumor burden. Three groups of 6 mice were
made according to tumor size one group with a tumor size of 40
mm.sup.2 (square), one with a tumor size of 50 mm.sup.2 (triangle)
and one with a tumor size of 100 mm.sup.2 (circle). At day 11 all
mice were injected intraperitoneally with 24 mg/kg of IP2-4Na. This
treatment was repeated 5 times every 3 to 4 days. In parallel,
tumor size was assessed every 3 to 4 days until reaching the
established ethical endpoints. Each line represents the tumor size
in area (mm.sup.2) of 6 mice in each group.
EXAMPLES
Materials & Methods
[0126] Cell culture
[0127] MCA205 mouse sarcoma cell line is cultured at 37.degree. C.
under 5% CO.sub.2 in RPMI 1640 medium (Life Technologies) in the
presence of 1% glutamine, 1% sodium pyruvate, 1% non-essential
amino-acids, 1% penicillin/streptomycin and 10% FBS (Life
Technologies) under standard conditions. B16F10 mouse melanoma cell
line, MRC5 human fibroblast cell line and A375 human melanoma cell
line are cultured at 37.degree. C. under 5% CO.sub.2 in DMEM medium
(Life Technologies) containing 1% glutamine, 1%
penicillin/streptomycin and 10% FCS under standard conditions. A549
human lung carcinoma cell line is cultured at 37.degree. C. under
5% CO.sub.2 in DMEM/F12+Glutamax I in the presence of 1% Hepes, 1%
sodium pyruvate and 10% FBS under standard conditions. Stable
MCA205-Globin-SL8-intron cell line are cultured under the same
condition as MCA205 cell line with additional 2 mg/ml G418
(geneticin from Life Technologies) for selection. Stable
B16F10-Globin-SL8-intron cell line are cultured under the same
condition as B16F10 cell line with additional 2 mg/ml G418
(geneticin from Life Technologies) for selection. The
SL8/Kb-specific (B3Z) T-cell reporter hybridoma are cultured at
37.degree. C. under 5% CO.sub.2 in RPMI 1640 medium (Life
Technologies) in the presence of 1% glutamine, 0.1%
.beta.-galactosidase, 1% penicillin/streptomycin and 10% FCS under
standard conditions.
[0128] T-Cell Assay
[0129] MCA205 and B16F10 mouse cell lines are transfected with the
plasmid YFP-Globin-SL8-intron or with the PCDNA3 empty plasmid
(negative control) with the transfection reagent jetPRIME (Ozyme)
or GeneJuice (Millipore) respectively according to each
manufacturer protocol. A375, A549 and MRC5 human cell lines are
transfected with the plasmid encoding mouse H2-Kb molecule for 12
hours followed by the transfection of the plasmid
YFP-Globin-SL8-intron with the transfection reagent jetPRIME
(Ozyme) according to the manufacturer protocol. Twenty-four hours
after transfection, cells are treated with different doses of
Isoginkgetin (Merk Millipore), IP2 or IM2P2 (also herein identified
as "M2P2") overnight. Then cells are washed three times with PBS 1X
and 5.times.10.sup.4 cells are co-cultured with 1.times.10.sup.5
B3Z cells. In positive control wells, 4 .mu.g/ml of synthetic
peptide SL8 is added. Cells are then incubated at 37.degree. C.
with 5% CO.sub.2 overnight. Cells are centrifuged at 1200 rpm for 5
min, washed twice with PBS 1X and lysed for 5 min at 4.degree. C.
under shaking with the following lysis buffer: 0.2% TritonX-100,
0.2% DTT, 0.5M K2HPO4, 0.5M KH2PO4. The lysate is centrifuged at
3000rpm for 10min and the supernatant is transferred to a 96-well
optiplate (Packard Bioscience, Randburg, SA). The revelation buffer
containing 33 mM of methylumbellifery .beta.-D-galactopyranoside
(MUG) is added and the plate is incubated at room temperature for 3
hours. Finally, the .beta.-galactosidase activity is measured using
the FLUOstar OPTIMA (BMG LABTECH Gmbh, Offenburg, Germany). Results
are expressed as mean .+-.SEM. *P<0.05, **P<0.01,
***P<0.001 (unpaired student t test).
[0130] Tumor Challenge and Treatment
[0131] C57B1/6J female mice are obtained from Harlan. NU/NU nude
mouse mice are obtained from Charles River. At 7 weeks old, mice
are injected subcutaneously on the right flank with
5.times.10.sup.4 MCA205-Globin-SL8-intron cells or with
4.times.10.sup.4 B16F10-Globin-SL8-intron cells along with matrigel
(VWR). Five days after challenge, mice are treated intraperitonealy
with PBS, Isoginkgetin (Merk Milllipore), IP2 or IM2P2. Fifteen
days after challenge mouse are again treated intreaperitonealy with
the same drug. Area of the tumor is recorded every 3 to 4 days
until day 27. All animal experiments were carried out in compliance
with French and European laws and regulations. Results are
expressed as mean .+-.SEM. *p<0.05, **P<0.01, ***P<0.001
(ANOVA with Tukey's multiple comparison test comparing all
groups).
Results
[0132] Isoginkgetin Treatment Increases Antigenic Presentation of
Intron-Derived Antigens in Cancer Cells.
[0133] In recent studies inventors have shown that Pioneer
Translation Products ("PTPs") are a major source of peptides for
the endogenous MHC class I pathway in vitro. In order to modulate
the presentation PTPs-derived antigens at cancer cells surface,
they tested the impact of isoginkgetin treatment on the melanoma
A375, the lung carcinoma A549 and on the normal fibroblast lung
MRC5 cell lines. For that purpose all the cells were transiently
expressing respectively the MHC class I K.sup.b molecule and the
SL8 epitope from an intron within the .beta.-Globin gene
constructs. As shown in FIGS. 1A, 1B and 1C, the natural
Isoginkgetin compound causes an increase in intron-PTPs-dependent
antigen presentation in cancer cell lines tested, with a dose
dependent effect. Similarly, the same experiment has been performed
on mice tumor cell lines, one melanoma (B16F10) and one sarcoma
(MCA205) cell lines. Both murine cell lines were transiently
expressing the PTPs-SL8 epitope derived from an intron within the
.beta.-Globin gene construct. Consistent with the previous results
in human cell lines, the Isoginkgetin elicits an increase in the
PTPs-dependent antigen presentation, with a dose dependent effect
in mouse cell lines. These results show that production and
presentation of PTPs antigens or PTPs-derived antigens can be
positively modulated in cancer cell lines upon isoginkgetin
treatment. They support the hypothesis that this molecule could be
used as positive immunomodulator to potentiate a specific
anti-tumoral immune response dependent on the PTPs production and
presentation.
[0134] Splicing Event is Required for an Efficient Increase of Exon
and Intron-Derived Antigenic Presentation in Cancer Cells after
Isoginkgetin Treatment.
[0135] The fact that Isoginkgetin increases the presentation of the
SL8 epitope at the cell surface from an intron encoded region
support the idea that pre-mRNAs are a source for antigen
presentation when the spliced machinery is unpaired. Inventors then
speculate that the Isoginkgetin will also elicit an increase of
antigenic epitope from an exon encoded region, but not from a cDNA
construct that does not need to be spliced. For that purpose, both
murine cell lines mentioned above were transiently expressing the
PTPs-SL8 epitope from an exon within the .beta.-Globin gene
construct or transiently expressing the Ova cDNA where the SL8
epitope is found in its right seeting. As expected, the natural
Isoginkgetin compound causes an increase in exon and
intron-PTPs-dependent antigen presentation in cancer cell lines
tested, with a dose dependent effect (FIGS. 2A, 2B, 2D and 2E),
whereas the splicing inhibitor has no effect on the production of
the SL8 epitope encoded by the Ova cDNA construct (FIGS. 2C and
2F). These results show that splicing is required for isoginkgetin
to act as a booster of the PTPs antigens or PTPs-derived antigenic
presentation in cancer cells.
[0136] Isoginkgetin treatment slows clown tumor growth in vivo.
[0137] The above results demonstrate that independently of the
source of PTPs-dependent antigen encoded by exon or intron
sequences, the natural product of Isoginkgetin is able to increase
their production and presentation in vitro at the cell surface of
treated tumor cell lines. The next evident question was to see if
the Isoginkgetin, which has to be dissolved in DMSO, can have the
same effect on tumor growth and CD8.sup.+ T cell proliferation in
vivo. For that purpose, MCA205 sarcoma cells stably expressing the
SIINFEKL (SL8) epitope from an intron in the .beta.-Globin gene
(Globin-intron-SL8) were subcutaneously inoculated in mouse. Five
days after this inoculation, the mice were intraperitoneally
vaccinated with a define dose of Isoginkgetin. Then, 10 days later
the same dose was again injected. During that time the tumor growth
was monitored every two to three days (FIG. 3A). Inventors observed
a significant 50% reduction of tumor growth at day 27 after
challenge in mice treated with 6, and 18 mg/kg of Isoginkgetin
(FIG. 3B). In order to assess the requirement of the immune
response for this effect, they tested the impact of 18 mg/kg
isoginkgetin treatment in immunodeficient nu/nu mice with the same
settings as previously described and observed that it has no effect
on the tumor growth (FIG. 4D). These results show that tumor size
reduction upon isoginkgetin treatment requires the presence of an
active immune response in vivo. Inventors next decided to generate
derivatives of the Isoginkgetin in order to try to increase the
anti-tumor response. In fact, isoginkgetin is insoluble in water
and can only be dissolved in DMSO solvant rendering its
pharmacokinetic in the peritoneal cavity less efficient.
[0138] Derivative Compounds from the Natural Isoginkgetin Product:
Schema of Synthesis.
[0139] In order to make isoginkgetin available for broader in vivo
validation without the use of toxic carriers or cosolvents (DMSO),
it was considered necessary to find a strategy to enhance its
solubility. The compounds of the invention were prepared from
commercially isoginkgetin, a small polyphenolic molecule more
commonly referred to as biflavonoid, extracted from leaves of
maidenhair tree, Ginko biloba L. Taking in account that the natural
products dissolved in DMSO might not be well assimilated in mouse,
inventors decided to generate derivative compounds that will have
kept their functions, meaning inhibitor of spliceosome and positive
immunomodulators against cancer cell lines with a better
pharmacokinetics than the natural compound dissolve in a
cosolvent.
[0140] The synthesis of IP2 compounds (2 and 2'), depicted in
Scheme 1 (FIG. 5), was accomplished from isoginkgetin by
phosphorylation employing in situ formation of
diethylchlorophosphite to provide 1. Further cleavage of the ethyl
ester protective groups with iodotrimethylsilane afforded the
phosphoric acid intermediate, which was immediately treated with
sodium hydroxide to complete a practical route to the disodium
phosphate prodrugs 2 and 2'. The water solubility of 2 and 2' was
found to be considerably higher than that of the parent compound
isoginkgetin.
[0141] The synthesis of IM2P2, derivative 4, was accomplished as
depicted in Scheme 1. The remaining two phenol groups of 1 were
alkylated using methyl iodide to furnish compound 3. Treatment of
this latter under similar conditions to prepare 2 and 2' from 1
gave the disodium phosphate prodrug 4, whereas its reaction under
basic conditions provided compound 5.
[0142] The prodrug 8 was synthesized in three steps from 6 by
phosphorylation of phenol groups in the C4 position followed by
cleavage of the ethyl groups of 7 with trimethylsilyl iodide and
reaction of the resulting phosphoric acid with sodium hydroxide in
water to afford the sodium phosphate prodrug 8.
##STR00007## ##STR00008##
[0143] Treatment of isoginkgetin with a large excess of methyl
iodide (5 equiv) under basic conditions furnished fully methylated
compound 11 [cf. Scheme 2 (FIG. 6)]. The use of 3 equivalents of
MeI produced a mixture of trialkylated products 9 and 10 which were
easily separated by column chromatography. Reaction of isoginkgetin
with pyridinium chloride allows ether cleavage and afforded the
polyphenolic compound 12.
##STR00009##
[0144] Isoginkgetin Derivative IP2 Efficiently Increases MHC Class
I Presentation of Intron-Derived Antigen in Vitro and Dramatically
Reduces Tumor Growth in vivo in an Immune-Dependent Manner.
[0145] In order to test the new compounds as positive
immunomodulators against tumor cell lines, inventors first decided
to test them in an in vitro assay. Both the derivatives 2 and/or
2', both also herein identified as "IP2", and the derivative 4,
called "IM2P2", were able to be dissolved in water. After treatment
of MCA205 cells transiently expressing PTPs-SL8 epitope derived
from an intron in the .beta.-Globin gene (Globin-intron-SL8) with
15 .mu.M, 25 .mu.M or 35 .mu.M of IP2, inventors observe an
increase in PTPs-dependent antigen presentation (FIG. 4A). On the
contrary, treatment of MCA205 cells expressing inventors'
PTPs-encoded construct with 15 .mu.M, 25 .mu.M and 35 .mu.M of
IM2P2 (FIG. 4B) or the product or compound 10 (FIG. 4C) does not
increase the presentation of inventors' PTPs-derived antigen. Then,
inventors decided to look in vivo at the effect of these
derivatives in term of anti-tumor growth and inducers of specific
anti-tumor immune responses. For that purpose, MCA205 sarcoma
cells, stably expressing the SIINFEKL (SL8) epitope from an intron
setting in the .beta.-Globin gene (Globin-intron-SL8), were
subcutaneously inoculated in mice. Then, 5 days after this
inoculation, each group of mice were respectively intraperitoneally
vaccinated with 18 mg/kg of Isoginkgetin, IP2 or IM2P2. Ten days
later, the same dose of each compound was again injected. During
that time the tumor growth was monitored every two to three days
(FIG. 3A). In the group of mice treated with 18 mg/kg of IP2,
inventors observed a dramatical decrease of tumor growth compared
to the group of mice treated with 18 mg/kg of Isoginkgetin (FIG.
4D). In parallel and on the contrary, mice that were treated with
18 mg/kg of IM2P2 did not demonstrate any tumor growth decrease
(FIG. 4D).
[0146] To demonstrate that the tumor growth decrease is due to
specific anti-tumor immune responses in a PTPs-dependent manner,
inventors looked at the effect of the compounds in Nu/Nu athymic
nude mice. For that purpose, MCA205 sarcoma cells stably expressing
the SIINFEKL (SL8) epitope from an intron setting in the
.beta.-Globin gene (Globin-intron-SL8) were subcutaneously
inoculated in mouse. Then, 5 days after this inoculation, each
group of mice were respectively intraperitoneally vaccinated with
18 mg/kg of Isoginkgetin, IP2 and IM2P2. Then 10 days later the
same dose of each compound was again injected. During that time the
tumor growth was monitored every two to three days (FIG. 3A). FIG.
4E shows that any of the compounds used have an effect on the tumor
growth of the sarcoma cell lines in these immunodeficient mice,
supporting and shedding further light on the fact that the selected
derivative of the natural product Isoginkgetin which is a splicing
inhibitor can be seen as positive immunomodulator against cancers
and can be used as a new chemotherapeutic treatment.
Discussion
[0147] The present invention demonstrates for the first time that
the product herein identified as IP2 has a very positive effect on
the antitumor immune response and therefore on tumor growth.
Inventors indeed demonstrated that a specific derivative of the
natural product Isoginkgetin is a potent stimulator of the
anti-tumor immune response in vitro and also in vivo. Such a small
molecule exhibiting this kind of mechanism of action is
unprecedented. Inventors' data open the way to new anti-cancer
applications within the framework of targeted molecular
therapies.
[0148] Pre-mRNA splicing is an essential mechanism required for the
normal function of all mammalian cells. In the last few years,
several studies reported the presence of mutations and
overexpression of main spliceosome factors associated with aberrant
splicing activity in various cancers. Few years ago, inventors have
also provided some evidence that the inhibition of the spliceosome
increases MHC class I PTPs-dependent antigen presentation. These
findings put the focus on the spliceosome as a potential target in
anti-cancer treatment. Small molecules have already been reported
to inhibit the spliceosome and specifically to inhibit the
spliceosome factor SF3B1 function. Although the precise mechanisms
of these small molecules are not yet completely understood, it has
been reported that they can be effective in cancer therapy by
reducing tumor size from 40 to 80% depending of the compound used.
The only one to date that has been tested in human is the E7107. It
has been stopped because of problems of toxicity. This compound is
known to inhibit the spliceosome by interacting with SF3B1. In that
study, inventors provide both in vitro and in vivo evidences that
by modulating the spliceosome activity using the herein described
specific derivative compound IP2 of the natural product
Isoginkgetin a specific anti-tumor immune response can be
induced.
[0149] Instead to look at the effect of these different compounds
inhibiting a specific component of the spliceosome inventors
decided to look at another class of inhibitors that have been
reported to inhibit the formation of the spliceosome complex.
Isoginkgetin has been reported to interfere in the early step of
assembly of the spliceosome and it has been also reported to be a
potent tumor cell invasion inhibitor. In fact, it has been
demonstrated that Isoginkgetin inhibits the A complex of the
pre-spliceosome to form a larger pre-catalytic spliceosome B
complex. Inventors demonstrated that by inhibiting the formation of
the spliceosome, as early as possible using derivative IP2 of the
Isoginkgetin in vitro and in vivo, the anti-tumor antigenic
presentation was increased very significantly, by inducing
specifically CD8.sup.+ T cell proliferations against PTPs-dependent
epitopes. Inventors herein report that IP2 can be used as a new
chemotherapeutic agent against cancer, in particular against
melanoma and sarcoma.
Additional Results
[0150] Splicing Inhibition Increases the Presentation of Antigens
Derived from Introns and Exons in Cancer Cells
[0151] Cancer cells display different intracellular mechanisms that
can shape the pool and the quantity of peptides presented on MHC
class I (MHC-I) molecules at their surface, leading to reduction of
their antigenicity and escape of T-cell recognition. Inventors have
shown that PTPs are a major source of peptides for the endogenous
MHC-I pathway in vitro. In addition, they have provided the first
proof of the positive impact of splicing inhibition on
PTPs-dependent antigen presentation by treating HEK cells with the
splicing inhibitor isoginkgetin. The latter has been reported to
inhibit the spliceosome during the early stages of its assembly. In
view to improve antigenicity and immune recognition of cancer cell
lines, inventors determined whether isoginkgetin was able to
modulate positively the expression and the presentation of tumor
associated PTPs-derived antigens (TA-PTPs) at cancer cells surface.
For that purpose, the human melanoma cell line A375, the human lung
cancer cell line A549 and the normal human fibroblast lung cell
line MRC5 were transiently expressing the MHC-I H2-K.sup.b molecule
and the PTPs-SL8 epitope from an intron within the .beta.-Globin
gene construct (Globin-SL8-intron) and treated with different
concentrations of isoginkgetin for 18 h. All results were expressed
based on the ratio of B3Z activation with and without extracellular
addition of the SL8 peptide in order not to be biased by
modulations in the overall expression of the H2-K.sup.b molecules
at the cell surface upon treatment. Treatment with isoginkgetin
increases intron-derived-SL8 antigen presentation in the three cell
types, in a dose dependent manner (FIG. 7A). Concentrations of
isoginkgetin used were not toxic for the human cells as the
viability is shown to be over 80% upon treatment (table 1).
TABLE-US-00001 TABLE 1 Percentage of human cell lines survival
after isoginkgetin treatment. % cell viability (.+-.SEM) Doses
(.mu.M) A375 A549 MRC5 Isoginkgetin 2.5 100 100 100 6.25 100 84
(.+-.1.86) 100
[0152] MTT assay on A375, A549 and MRC5 cell lines treated with 2.5
.mu.M or 6.25 .mu.M of isoginkgetin. Dare are given as the mean of
the percentage of cell viability .+-.SEM from at least three
independent experiments.
[0153] In parallel, the same experiment was performed on the mouse
melanoma B16F10 and sarcoma MCA205 cell lines that were transiently
expressing the Globin-SL8-intron construct. Consistent with the
previous results, the isoginkgetin elicits an increase in the
intron-derived-SL8 antigen presentation, in a dose dependent manner
(FIG. 7B). Efficient doses lead to cell viability over 50% in these
cell lines (table 2).
TABLE-US-00002 TABLE 2 Percentage of human cell lines survival
after isoginkgetin and treatment. % cell viability (.+-.SEM) Doses
(.mu.M) MCA205 B16F10 Insoginkgetin 6.25 84.8 (.+-.9.6) 86.5
(.+-.2.2) 15 56.3 (.+-.4.9) 58.6 (.+-.3.8) 25 51.7 (.+-.4.2) 51.2
(.+-.4.8)
[0154] MTT assay on MCA205 and B16F10 cell lines treated with 6.25
.mu.M, 15 .mu.M or 25 .mu.M of isoginkgetin. Dare are given as the
mean of the percentage of cell viability .+-.SEM from at least
three independent experiments.
[0155] To investigate further the impact of isoginkgetin on PTPs
presentation, both murine sarcoma and melanoma cell lines were
transiently expressing the PTPs-SL8 epitope from an exon within the
.beta.-Globin gene construct (Globin-SL8-exon) or transiently
expressing the Ova cDNA. In the latter construct, the SL8 epitope
is found in its right setting and does not need to be spliced.
Inventors observed that the isoginkgetin increases exon-derived-SL8
antigen presentation in the MCA205 and in the B16F10 cancer cell
lines with a dose-dependent effect (FIG. 7C), whereas the splicing
inhibitor has no effect on the production of the SL8 epitope
encoded by the Ova cDNA construct (FIG. 7D). Hence, splicing event
seems to be required for isoginkgetin to impact PTPs-dependent
antigen presentation. This suggests an action of isoginkgetin
during the production step of PTPs and not further down in the
MHC-I antigen presentation pathway. Along with these results,
inventors showed that the expression of the H2-K.sup.b molecules at
the cell surface is affected differently in the cell lines treated
with isoginkgetin, i.e. it decreases in MCA205 (FIG. 8A, left
panel), increases in B16F10 (FIG. 8A, right panel) and is stable on
human cell lines (FIG. 8B).
[0156] Overall, these results show that the natural product
isoginkgetin acts as a booster of the PTPs-derived antigen
presentation in cancer cells independently of the epitope setting,
i.e. in exonic or in intronic sequences, and independently of the
cell lines tested. This shed light on the importance of the
splicing event for the production and the presentation of MHC-I
antigens in cancer cells. Finally, inventors' data support the idea
that pre-mRNAs are a source for antigen presentation when the
spliced machinery is unpaired.
[0157] Isoginkgetin Treatment Slows Clown Tumor Growth in vivo when
the Intron-Derived-SL8 Epitope is Expressed and its action is
Dependent on the Immune Response.
[0158] Antigens abundance at the cell surface has been demonstrated
to be a key parameter in determining the magnitude of the CD8.sup.+
T cell response and hence in defining immunodominance (Doherty et
al., 2006). The SL8 peptide has been widely shown to be highly
immunogenic in vivo. Looking at SL8-specific T-cells activation in
vitro, inventors observed a change in the abundance of the SL8
expression at the cancer cells surface after splicing inhibition.
In order to test this hypothesis in vivo, they first looked at the
impact of isoginkgetin treatment on the growth of tumors that
express the intron-derived SL8 peptide. For that purpose, both
MCA205 sarcoma cells and B16F10 melanoma cells that stably express
the globin-intron-SL8 construct were inoculated subcutaneously in
mice. At days 5, 10 and 15 after tumor inoculation, the mice were
injected intraperitoneally with a define dose of isoginkgetin, and
the tumor growth was monitored (FIG. 9A). In mice bearing MCA205
globin-SL8-intron (MCA205 GI) tumors, inventors observed a
significant reduction of tumor size, over 50% at day 27 after
challenge when treated with 12 and 18 mg/kg of isoginkgetin (FIG.
9B). The impact of isoginkgetin treatment on B16F10
globin-SL8-intron (B16F10 GI) tumor growth is lower than on MCA205
GI; however the drug still significantly slows down tumor growth
(FIG. 9C). To assess the link between SL8 overexpression and tumor
growth reduction in vivo after isoginkgetin treatment, inventors
performed the same experiment with mice inoculated with either
MCA205 or B16F10 wild type (WT) cells. No significant reduction of
MCA205 WT (FIG. 9D) or B16F10 WT (FIG. 9E) tumor growth was
observed after treatment with 12 and 18 mg/kg of isoginkgetin.
These results suggest that the expression of an immunodominant
epitope, herein the SL8 peptide, is required for isoginkgetin to
impact tumor growth in vivo.
[0159] Inventors then assessed the requirement of the immune
response for isoginkgetin to reduce tumor growth Immunodeficient
Nu/Nu nude mice were inoculated subcutaneously with either MCA205
or B16F10 cells that stably express the Globin-intron-SL8 or WT,
and were treated with the same settings as previously described
(FIG. 9A). No effect of isoginkgetin treatment was observed on the
growth of each of the four tumor types (FIG. 9F-I).
[0160] Overall, these results show that tumor size reduction upon
isoginkgetin treatment requires the presence of an active immune
response in vivo, and suggest that the increase of the expression
of an immunodominant epitope drives the anti-tumor immune
response.
[0161] The Compound Derivatives from the Natural Isoginkgetin
(Product IP2 and Product M2P2, the Last One Being also Herein Above
Identified as "IM2P2") are Water Soluble, Inhibit the Splicing and
are Less Toxic.
[0162] Derivatives of the natural isoginkgetin product were
synthesized and tested for their ability to inhibit the splicing,
to increase PTPs-derived antigens in vitro as well as for their
ability to reduce tumor growth in vivo. The derivatives IP2 (see
IP2-6Na and IP2-4Na on FIG. 10B) and M2P2 (FIG. 10C) were
synthesized from the commercial isoginkgetin (FIG. 10A), more
commonly referred to as biflavonoid, extracted from leaves of
maidenhair tree, Ginko biloba L. Schema of synthesis is provided in
FIG. 5. Briefly, the synthesis of IP2 or compounds 2 and 2' (as
named in the schema) was accomplished by the phosphorylation of
isoginkgetin employing in situ formation of diethylchlorophosphite
to provide compound 1. Further cleavage of the ethyl ester
protective groups with iodotrimethylsilane afforded the phosphoric
acid intermediate, which was immediately treated with sodium
hydroxide to complete a practical route to the disodium phosphate
prodrug. For the synthesis of the M2P2 molecule, the remaining two
phenol groups of compound 1 were alkylated using methyl iodide to
furnish compound 3. Treatment of the latter under similar
conditions to prepare compounds 2 and 2' from compound 1 gave the
disodium phosphate prodrug 4 or M2P2, whereas its reaction under
basic conditions provided compound 5.
[0163] The water solubility of IP2 and M2P2 was found to be
considerably higher than that of the parent compound isoginkgetin
(data not shown). In addition, inventors tested the ability of IP2
and M2P2 to inhibit the splicing of the Globin-SL8-intron gene
product in MCA205 and B16F10 cells. Interestingly, IP2 and M2P2
provide two distinct patterns of splicing inhibition in each cell
line. IP2 treatment increases the presence of non-spliced RNA
products in both cells, such as isoginkgetin treatment does. In
contrast, M2P2 treatment does not impact splicing in B16F10 cells
while it has a strong impact on splicing in MCA205 compared to IP2
and isoginkgetin treatment (FIG. 10D and 10E). Hence, IP2 and M2P2
appear to display different mechanisms for inhibiting the splicing.
Importantly, inventors observed that the splicing pattern of IP2 is
similar to the one of isoginkgetin for the studied gene product. In
addition, it has been shown that cancer cells can acquire
deficiencies in the splicing machinery that benefit their growth,
for example by preventing the expression of tumor suppressor genes.
These deficiencies do not have the same nature in all tumors and
therefore could explain the distinct effects of splicing inhibitors
on separate tumor types. Both IP2 and M2P2 display no toxicity in
MCA205 and B16F10 WT cells at the doses tested (FIG. 10F and 10G).
Overall, inventors have provided and herein identify for the first
time two new drugs that are water soluble and that can impact the
splicing differently in two distinct model cell lines at doses that
do not impact cells viability.
[0164] Isoginkgetin Derivative IP2 Efficiently Increases MHC-I
Presentation of Intron-Derived Antigen in Vitro, Reduces Tumor
Growth in vivo and Extends Survival.
[0165] In order to test the potential immunomodulatory effect of
IP2 and M2P2 compounds in comparison with isoginkgetin, the two
molecules were first tested for their ability to increase the MHC-I
presentation of PTPs-derived antigens in vitro. For that purpose,
MCA205 and B16F10 cells were transiently expressing the
Globin-intron-SL8 construct and treated with 15 .mu.M or 35 .mu.M
of IP2 or M2P2. While treatment with IP2 increases the
intron-SL8-derived antigen presentation in MCA205 and B16F10 cells
similarly to what inventors observed after isoginkgetin treatment
(FIG. 11A and B, left panels), M2P2 decreases its presentation in
MCA205 cells and does not impact it in B16F10 cells (FIG. 11A and B
right panels). These results are interestingly correlated to the
respective ability of IP2 and M2P2 to inhibit the Globin-SL8-intron
gene splicing. In fact, M2P2 has no impact both on splicing and on
the SL8 antigen presentation in B16F10. Conversely, M2P2 strongly
inhibits the splicing in MCA205 and negatively affects the SL8
presentation. Along with these results, inventors showed that the
expression of the H2-K.sup.b molecules at the cell surface is not
affected in the cell lines treated with IP2 and M2P2 (FIG. 12A and
12B). Hence, it seems likely that a tight regulation of splicing is
required for treatments to positively impact the presentation of
intron-derived epitopes. These results show that the isoginkgetin
derivative IP2 acts as a booster of the PTPs-derived antigenic
presentation in vitro in the same way as the natural product.
[0166] The IP2 and M2P2 molecules were then tested for their
anti-tumoral effect in vivo. As previously performed with
isoginkgetin treatment, MCA205 sarcoma cells or B16F10 melanoma
cells, stably expressing the Globin-intron-SL8 construct or WT,
were subcutaneously inoculated in mice. At days 5, 10 and 15 after
tumor inoculation, each group of mice were respectively
intraperitoneally treated with 18 mg/kg of isoginkgetin, IP2 or
M2P2. At this dose, a significant decrease of MCA205 GI tumor
growth was observed after treatment with IP2 compare to
isoginkgetin treatment, while no impact of M2P2 treatment was
monitored (FIG. 11C, left panel). In addition, the reduction of
B16F10 GI tumor growth was similar after treatment with 18 mg/kg of
isoginkgetin or IP2, while M2P2 had no effect on growth (FIG. 11C,
right panel). As IP2 treatment reduces tumor growth and is water
soluble, inventors decided to increase the dose injected in mice.
The increased dose of IP2 did not improve its anti-tumoral effect
on MCA205 GI at 24 mg/kg but increased it on B16F10 GI at 36 mg/kg
(FIG. 11C). Strikingly, isoginkgetin and M2P2 treatment did not
impact tumor growth of either MCA205 WT or B16F10 WT, whereas IP2
treatment slows down both tumor growth (FIG. 11D). Inventors
confirmed that IP2 does not induce apoptosis of tumor cells even at
a high dose (FIG. 12C). Finally, IP2 treatment was shown to extend
survival of mice, with more than 50% of survivors 100 days after
tumor inoculation (FIG. 11E, lower panel). At the same time, around
30% of mice treated with isoginkgetin were still alive (FIG. 11E,
middle panel). M2P2 treatment does not impact survival (FIG. 11E,
upper panel).
[0167] Overall, these results demonstrate a correlation between an
increase of PTPs-derived antigen presentation observed in vitro and
a reduction of the tumor growth in vivo after treatment.
Interestingly, contrary to isoginkgetin, IP2 treatment slows down
the growth of tumors that do not bear the highly immunodominant SL8
epitope derived from PTPs. Inventors believe that the splicing
inhibitor IP2 potentiates the apparition of immunodominant epitopes
at the cell surface that drives the anti-tumoral response.
[0168] IP2 Treatment Efficacy is Dependent on the Immune-Response
and Creates a Long-Lasting Anti-Tumoral Response.
[0169] To determine the requirement of the immune system and
especially of the T-cell response for IP2 efficacy against tumor,
inventors looked at its effect in Nu/Nu athymic nude mice that lack
T-cells but not B and NK cells. As previously tested with
isoginkgetin, MCA205 or B16F10 cells stably expressing the
Globin-intron-SL8 construct or WT were subcutaneously inoculated in
mice. At days 5, 10 and 15 after tumor inoculation, each group of
mice were intraperitoneally treated with the most efficient dose of
IP2 against tumor growth observed in immunocompetent mice. Hence,
MCA205GI, MCA205 WT and B16F10 GI or WT bearing mice were treated
with 18 mg/kg, 24 mg/kg and 36 mg/kg respectively. In each
condition, no impact of IP2 treatment was observed on tumor growth
(FIG. 13A and B).
[0170] In addition, in order to assess the specific requirement of
CD8.sup.+ T cells for IP2 efficacy, inventors tested the impact of
in vivo CD8.sup.+ T cells depletion in mice. Mice were inoculated
with MCA205 GI cells subcutaneously followed by a scheduled
treatment with anti-CD8.sup.+ T cells antibody or with the isotype
2A3. IP2 treatment was administered as previously at day 5, 10 and
15. Interestingly, anti-CD8.sup.+ T cells antibody treatment
completely abrogated the anti-tumoral effect of the IP2 treatment
(FIG. 13C). Therefore, this result confirms that the effect of the
IP2 treatment on tumor growth is dependent on the CD8.sup.+ T cell
response, which supports an antigen-driven cytotoxic activity
against the tumor cells.
[0171] Finally, around 50% of mice inoculated with MCA205 GI and
subsequently treated with IP2 as described above became tumor free
after treatment. 100 days after the first tumor inoculation, these
mice were re-challenged with MCA205 GI tumor cells on the right
flank and with B16F10 cells on the left flank. While B16F10 tumors
grew over time, MCA205 GI did not grow in mice (FIG. 13D). These
results demonstrate that mice developed a long term anti-tumoral
response specific to MCA205 GI tumor after IP2 treatment.
[0172] IP2 (IP2-4Na) Treatment Reduces Tumor Growth from
Established Tumor
[0173] In order to assess the specific IP2 efficacy on modulating
tumor growth by enhancing the proliferation of specific anti-tumor
CD8.sup.+ T cells, inventors tested the impact of IP2 on
established tumor in vivo. MCA205 sarcoma tumor cells (15.10.sup.5)
stably expressing the Globin-intron-SL8 construct were injected
subcutaneously and were allowed to progress 10 days before being
ranked and assigned to groups of equivalent tumor burden, resulting
in the formation of three groups of tumor sizes of 40, 50 and 100
mm.sup.2 before initiation of IP2 treatment. Then every 3 to 4
days, each group of mice was respectively intraperitoneally treated
with 24 mg/kg of IP2. At this dose, a remarkable decrease of MCA205
GI tumor growth was observed after treatment with IP2 compare to
non-treated mice (FIG. 14) independently of the group tested. Even
when the tumors had reached a size of 100 mm.sup.2 (circle), 5
doses of 24 mg/kg of IP2 were able to induce a significant tumor
regression with an increase of survival of around 30 days (grey
circle) compare to the untreated mice (black circles). In
conclusion, all these experiments using IP2 as immunomodulators
show that this molecule is efficient to induce a complete tumor
rejection when mice are treated very early (when tumor where just
palpable) by inducing a long term anti-tumoral response, and show
that IP2 treatment can also reduce tumor growth on established
tumor.
[0174] Overall these results shed light on the capacity of specific
splicing inhibitors, such as isoginkgetin and IP2, to positively
modulate the anti-tumoral immune response. In addition, they
confirm that PTPs-derived antigens are efficiently presented and
recognized by CD8.sup.+ T cells in vitro and in vivo and that a
change in their presentation at the cell surface in quantity or in
quality can lead to a CD8.sup.+T cells response against cancer.
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Sequence CWU 1
1
118PRTArtificial SequenceSEQ01 1Ser Ile Ile Asn Phe Glu Lys Leu1
5
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